Dean W. Felsher - US grants

Cancer is caused by the progressive accumulation of multiple genetic abnormalities in proto-oncogenes and tumor suppressor genes (1). The appreciation that specific cancers are associated with discrete combinations of genetic events suggests that a highly effective and specific form of cancer therapy would be to target the repair, replacement or inactivation of oncogenes or tumor suppressor genes (1). Nevertheless, this assumes both that cancer as a multi-step process is generally reversible and that cancer cells will not be able to compensate for the repair of a single abnormal genetic event. The C-MYC proto-oncogene is commonly activated in specific human neoplasia and may be an excellent target for the therapy of cancer. The goal of the proposed research is to determine if the targeted inactivation of C-MYC will be effective as a treatment for cancer. Preliminary data described in this proposal demonstrate that C-MYC induced tumorigenesis of the immortal cell line Rat-1a is not reversible with inactivation of C-MYC. Perhaps accounting for this observation, Rat-1a cells become rapidly genomically unstable with C-MYC activation. Experiments will be performed to determine if C-MYC induces tumorigenesis reversible with C-MYC inactivation in other rodent and human cell lines and in transgenic mice, to determine if C-MYC activation generally induces genomic instability, to determine if other oncogenes, genes transcriptionally regulated by C-MYC, and genes that are associated with acceleration with C-MYC tumorigenesis induce genomic instability, and to determine in cases where tumors persist despite C-MYC inactivation, the genetic events responsible for a persistent malignant phenotype. If C-MYC activation generally induces tumorigenesis that is reversed with C-MYC inactivation, then it will be concluded that C-MYC inactivation is likely to be an effective therapy for C-MYC induced cancers. If this is not the case, the genetic events responsible for a persistent tumorigenic phenotype will be determined. The targeted the repair of these genetic events may be more likely to be effective in the treatment of C-MYC induced human cancers. The results obtained from these studies will not only provide a better understanding of the mechanism of C-MYC induced tumorigenesis and C-MYC induced genomic instability, but will identify the best targets for the therapy of C-MYC induced cancers.

DESCRIPTION: (provided by applicant) The targeted repair or inactivation of damaged proto-oncogenes (oncogenes) may be a specific and effective treatment for neoplasia. My long-term goal is to understand bow oncogenes induce and maintain tumorigenesis. My strategy has been to generate a conditional transgenic model system using the tetracycline regulatory system to investigate how oncogene activation causes tumorigenesis and when continued activation is required to maintain a tumorigenic phenotype. I have focused on investigating how over-expression of the MYC proto-oncogene causes tumorigenesis. My hypothesis is that the mechanism by which MYC induces tumorigenesis will define when its continued expression is required to maintain a tumorigenic phenotype, and thus, when its inactivation will induce tumor regression. Recently I have shown that MYC-induced tumors regressed upon MYC inactivation (see Appendix, Feisher and Bishop, Molecular Cell, 1999). From these results, I conclude that there are circumstances when the inactivation of MYC can cause tumor regression. Now, I propose experiments to address the following three specific aims:(1) 1 will determine how MYC activation induces tumorigenesis in hematopoietic cells by influencing the cell cycle, genomic stability and apoptosis and how other oncogenes (p53-I-, p]9ARF-/-, ROAS, BCL2) cooperate with MYC to influence these same parameters. (2) I will determine how MYC inactivation causes tumor regression, if MYC re-activation permits tumor relapse, and if cooperating oncogenic events (p53-I-, p19ARF-/-, RAS, BCL2 ) prevent regression or promote relapse. (3) I will determine how MYC maintains a tumorigenic phenotype by examining if (MYC family members (N-, L-MYC), S-MYC or MYC mutants defective for specific functional domains or MYC 's transcriptional targets (ODC, eIF-4E, TERT) can functionally replace MYC. I will attempt to identify novel genes that can functionally replace MYC to maintain in part or whole its neoplastic phenotype. The results obtained from these studies will be useful in determining how MYC causes tumorigenesis and defining when the inactivation of MYC is likely to be effective in the treatment of human neoplasia.

DESCRIPTION (provided by applicant): The MYC oncogene is a transcription factor whose overexpression is thought to induce tumorigenesis by causing inappropriate gene expression that results in autonomous cellular proliferation and a block in cellular differentiation. Recently, we have found that even the brief inactivation of the MYC oncogene can result in the sustained loss of a neoplastic phenotype. We demonstrated that upon MYC inactivation immature osteogenic sarcoma cells differentiated into mature osteocytes and formed bone. Surprisingly, MYC reactivation failed to restore a neoplastic phenotype and instead induced the now differentiated tumor cells to undergo apoptosis. Our results suggest to us the interesting possibility that MYC inactivation causes the loss of a neoplastic phenotype by inducing tumors to differentiate. In this new epigenetic context, the ability of the MYC oncogene to induce the expression of genes responsible for sustaining cellular proliferation and hence tumorigenesis has been revoked and now instead apoptosis occurs. We hypothesize, that upon differentiation the tumor cells undergo chromatin remodeling and this new epigenetic program prevents MYC from inducing the transcription of genes required to induce or sustain tumorigenesis. Instead, MYC activation induces apoptosis. To examine for this possibility, we propose: to determine the duration of MYC inactivation required to result in sustained loss of a neoplastic phenotype by time lapsed video microscopy; to determine if the ability of MYC to activate the transcription of different gene targets changes upon differentiation of tumor cells by examining the expression of specific target genes by RT-PCR and global changes in gene expression by cDNA microarrays and by measuring the accessibility of specific target gene loci to MYC binding by chromatin immunoprecipitation and histone acetylation; and to determine if the ability of MYC to induce apoptosis changes upon the differentiation of tumor cells by examining the change in expression patterns of both pro- and anti-apoptotic gene products and by directly determining the effects of the inhibition of apoptotic pathways. The results of our experiments may have important implications for how the state of differentiation of a cell influences the ability of MYC to induce gene expression and thereby initiate and sustain tumorigenesis.

Cancer is a multi-step process caused by genetic abnormalities in proto-oncogenes and tumor suppressor genes. A specific and effective form of cancer therapy may be to target the repair, replacement or inactivation of oncogenes. The ability of oncogene activation to induce tumorigenesis and inactivation to induce tumor regression are likely to depend on many parameters including the constellation of genetic events, epigenetic factors, as well as host factors such as immune mechanisms. Previously, we have demonstrated that oncogene-induced tumorigenesis is reversible utilizing conditional transgenic model systems that employ the Tet system. We have developed transgenic models in which we can conditionally regulate MYC, RAS and/or BCL2 oncogenes. Now, we will employ these models systems to examine how genetic context and host immune status influences the ability of oncogene activation to induce tumorigenesis and inactivation to reverse tumorigenesis. A key difficulty in interrogating these processes is the inability to directly follow specific pre-neoplastic and neoplastic subsets of cells within the context of a living subject. Similarly, it would be desirable to examine the consequences of the inactivation of specific oncogenic events within an established tumor. Logistically, it is impossible to perform a kinetic analysis of tumorigenesis through the examination of the multitude of mice required. Analytically, it is not possible to examine the precise consequences of oncogene activation and inactivation ex vivo - the ability to examine specific biologic outcomes in situ permits the evaluation of host context. The use of combined imaging including Bioluminescence Imaging (BLI), microPET and Flow Cytometry simultaneously will permit us to interrogate effects on multiple biological outcomes such as proliferative expansion, apoptosis, and intracellular signaling. Preliminary results will be presented that demonstrate that BLI provides a unique and powerful approach to gain new insight into how and to what extent the activation of oncogenes can induce tumorigenesis, as well as how oncogene inactivation induces the regression of hematopoietic tumors. We now propose to utilize BLI to analyze the kinetics of tumor regression upon MYC inactivation, in conjunction with microPET to analyze angiogenesis and phosphorylation state of MYC and phosphoprotein FACS analysis to examine associated changes in phosphoprotein expression. Through multi-modality imaging, we will gain insight into the mechanisms by which oncogenes initiate and sustain tumorigenesis that will serve as important clues towards the development of therapies that target oncogenes for the treatment of cancer.

Cancer is a multi-step process caused by genetic abnormalities in proto-oncogenes and tumor suppressor[unreadable] genes. A highly specific and effective form of cancer therapy may be to target the repair, replacement or[unreadable] inactivation of oncogenes. Indeed, we and others have shown that the inactivation of a single oncogene can[unreadable] induce sustained tumor regression. However, we have also shown that tumors can escape dependence[unreadable] upon oncogenes. Now, to better interrogate the mechanisms by which oncogenes initiate and sustain[unreadable] tumorigenesis, we have generated a transgenic model in which we can temporally regulate, alone or in[unreadable] combination the expression of oncogenes, to study in vivo the kinetics of oncogene induced tumor[unreadable] regression upon oncogene inactivation. We have begun to use this strategy to examine how oncogene[unreadable] inactivation induces tumor regression, the role of host immune mechanisms to mediate this tumor regression[unreadable] and the genetic mechanisms by which cancers escape dependence upon oncogenes. We have obtained[unreadable] several potentially important preliminary results. First, we have found that when tumors are transplanted into[unreadable] immune compromised hosts, oncogene inactivation is much less capable of inducing tumor regression.[unreadable] These results suggest that immune mechanisms play a key role in mediating tumor regression upon[unreadable] oncogene inactivation. Second, we have used Spectral Karyotypic Analysis (SKY) and CGH Microarrays to[unreadable] demonstrate that tumors that escape dependence upon oncogenes have acquired novel chromosomal[unreadable] translocations. Our expectation is that both cell extrinsic and intrinsic mechanisms are involved tumor[unreadable] escape from oncogene dependence. In some cases, these mechanisms may converge. Thus, there are[unreadable] likely to be genetic events that impair the response of tumor cells to a host mediated suppression of[unreadable] tumorigenesis.

Cancer is a multi-step process caused by genetic abnormalities in proto-oncogenes and tumor suppressor genes. A highly specific and effective form of cancer therapy may be to target the repair, replacement or inactivation of oncogenes. Indeed, we and others have shown that the inactivation of a single oncogene can induce sustained tumor regression. However, we have also shown that tumors can escape dependence upon oncogenes. Now, to better interrogate the mechanisms by which oncogenes initiate and sustain tumorigenesis, we have generated a transgenic model in which we can temporally regulate, alone or in combination the expression of oncogenes, to study in vivo the kinetics of oncogene induced tumor regression upon oncogene inactivation. We have begun to use this strategy to examine how oncogene inactivation induces tumor regression, the role of host immune mechanisms to mediate this tumor regression and the genetic mechanisms by which cancers escape dependence upon oncogenes. We have obtained several potentially important preliminary results. First, we have found that when tumors are transplanted into immune compromised hosts, oncogene inactivation is much less capable of inducing tumor regression. These results suggest that immune mechanisms play a key role in mediating tumor regression upon oncogene inactivation. Second, we have used Spectral Karyotypic Analysis (SKY) and CGH Microarrays to demonstrate that tumors that escape dependence upon oncogenes have acquired novel chromosomal translocations. Our expectation is that both cell extrinsic and intrinsic mechanisms are involved tumor escape from oncogene dependence. In some cases, these mechanisms may converge. Thus, there are likely to be genetic events that impair the response of tumor cells to a host mediated suppression of tumorigenesis.

Cancer is a multi-step process caused by genetic abnormalities in proto-oncogenes and tumor suppressor genes. A specific and effective form of cancer therapy may be to target the repair, replacement or inactivation of oncogenes. The ability of oncogene activation to induce tumorigenesis and inactivation to induce tumor regression are likely to depend on many parameters including the constellation of genetic events, epigenetic factors, as well as host factors such as immune mechanisms. Previously, we have demonstrated that oncogene-induced tumorigenesis is reversible utilizing conditional transgenic model systems that employ the Tet system. We have developed transgenic models in which we can conditionally regulate MYC, RAS and/or BCL2 oncogenes. Now, we will employ these models systems to examine how genetic context and host immune status influences the ability of oncogene activation to induce tumorigenesis and inactivation to reverse tumorigenesis. A key difficulty in interrogating these processes is the inability to directly follow specific pre-neoplastic and neoplastic subsets of cells within the context of a living subject. Similarly, it would be desirable to examine the consequences of the inactivation of specific oncogenic events within an established tumor. Logistically, it is impossible to perform a kinetic analysis of tumorigenesis through the examination of the multitude of mice required. Analytically, it is not possible to examine the precise consequences of oncogene activation and inactivation ex vivo - the ability to examine specific biologic outcomes in situ permits the evaluation of host context. The use of combined imaging including Bioluminescence Imaging (BLI), microPET and Flow Cytometry simultaneously will permit us to interrogate effects on multiple biological outcomes such as proliferative expansion, apoptosis, and intracellular signaling. Preliminary results will be presented that demonstrate that BLI provides a unique and powerful approach to gain new insight into how and to what extent the activation of oncogenes can induce tumorigenesis, as well as how oncogene inactivation induces the regression of hematopoietic tumors. We now propose to utilize BLI to analyze the kinetics of tumor regression upon MYC inactivation, in conjunction with microPET to analyze angiogenesis and phosphorylation state of MYC and phosphoprotein FACS analysis to examine associated changes in phosphoprotein expression. Through multi-modality imaging, we will gain insight into the mechanisms by which oncogenes initiate and sustain tumorigenesis that will serve as important clues towards the development of therapies that target oncogenes for the treatment of cancer.

[unreadable] DESCRIPTION (provided by applicant): This proposal is in response to RFA PAR-07-105: Shared Instrumentation Grant Program. Stanford has been a leader in the development of new technologies for the analysis of the genome and proteome including pioneering the development of recombinant DNA, the discovery of flow cytometry and the design of DNA, RNA and protein microarrays. The aim of this proposal is to further build upon Stanford's outstanding Immunology and Cancer Center Core Facilities programs that are integral to basic, clinical and translational research programs by providing a novel, state of the art technology for performing nano-scale proteonomic analysis. Firefly is a fully automated, rapid quantitative protein analysis apparatus that incorporates isoelectric focusing with antibody detection to measure specific protein abundance and modification. This new method provides unparalleled ability to bring quantitative protein analysis for the dissection of complex biological problems as well as the examination of human clinical specimens. In this proposal, two major themes should be apparent. First, that the intention of this grant is to make Firefly available to a large community of investigators that have a mutual interest in the development of nano-scale proteonomic analysis in the fields of Immunology and and/or Cancer Biology; and with a commitment to scientific and conceptual interactions amongst basic scientists and physician scientists. [unreadable] [unreadable] Relevance: our projects will integrate an understanding of the mechanism of action of therapeutics [Felsher, Robinson] with new mechanistic insight into pathogenesis of disease states [Herzenberg, Steinman, Nolan, Mitchell], characterize the role of stem cells in diseases [Jeffrey, Chang, Sage] and develop novel diagnostic tests [Felsher, Robinson]. Our work involves the analysis of clinical material from preclinical animal models [Felsher, Sage] and human patients [Felsher, Mitchell, Jeffrey]. Our proposal thus thematically resonates with the greater vision of our Dean of Medicine [see letter, Dean Pizzo], who has reorganized Stanford around broad themes that integrate basic and clinical sciences around institutes that encompass immunology, cancer biology, neurosciences and cardiopulmonary biology. Our proposal will bring together two of these research communities: Immunology and Cancer Biology with guaranteed support of from the Stanford Immunology Transplantation and Infectious Disease Institute [see letter, Mark Davis] and the Cancer Center [see letter, Beverly Mitchell] through the Immune Monitoring Core [IMC], as directed by Dr. David Hirschberg. A vital additional component of our program involves the integration of data through support from Bioinformatics [see Letter, Amar Das]. [unreadable] [unreadable] [unreadable]

Cancer is caused by the activation of oncogenes and the inactivation of tumor suppressor genes. The targeted inactivation and repair of these gene products may be a specific and effective therapy for cancer. Previously, we have shown that the inactivation of the MYC oncogene is sufficient to induce sustained regression of hematopoietic tumors [2]. Subsequently, we reported that even brief inactivation of MYC is sufficient to induce sustained tumor regression of osteosarcoma [3, 4]. Our results support the general hypothesis that tumors exhibit the phenomena of oncogene addiction [5, 6]. To explain our findings, we reasoned that MYC inactivation induces a permanent change in the ability of cells to induce a cancer-associated gene expressionprogram. Upon MYC inactivation, there are specific and sustained changes in gene expression [Wu et al, PLoS Genetics [1];and see Appendix, Shachaf et al, Cancer Research, 2008]. These changes in gene expression are frequently accompanied by permanent changes in the ability of MYC binding to promoter loci, as shown by ChIP. We performed a preliminary ChIP-on-chip analysis for MYC and interrogated changes in binding of other transcription factors in a genome-wide scale. Moreover, we also found that changes in gene expression are associated with specific alterations in chromatin modifications. Importantly, MYC appears to regulate gene expression not just through interactions with the canonical DNA binding sequence (E-Box), but additionally, its binding specificity may be regulated by DNA methylation. We provide new results that illustrate that the TGF-n signaling pathway may play an important role in the mechanism by which MYC inactivation induces changes in gene expression and cellular senescence. Moreover, we provide evidence that MYC regulates DNA methylation. Hence, we hypothesize that MYC inactivation restores auto-regulatory programs, including the induction of CDKIs, through effects on DNA methylation, resulting in the induction of a cellular senescence program. Hence, both cell extrinsic receptor based mechanisms, including TGF-U signaling, as well as cell intrinsic mechanisms, including regulation of DNA methylation, may be critical to oncogene addiction. We now propose experiments and request salary and grant support for a team of 1 principal investigator, 2 post-doctoral fellows and 1 graduate student and a research associates to delineate the role of TGF-13 signaling and DNA methylation on the mechanism of tumor regression upon MYC inactivation. Our results are consistent with the notion that MYC may directly regulate the global chromatin structure;and suggest the surprising idea that MYC-induced tumor cells remain unaware or "amnesic" - as we have recently described [10] - of their cancerous state, yet remain poised to undergo senescence. The results of our proposed experiments will have important implications for the mechanisms by which the MYC oncogene maintains tumorigenesis and the development of new therapies for the treatment of cancer.

Transgenic mouse models provide the possibility to study cancer progression in vivo in a controlled, experimentally manipulable manner. The Myc oncogene is deregulated in a wide range of human cancers, and its activity is often associated with highly aggressive tumor behavior. Myc is a transcription factor that regulates the expression of several thousand genes both directly and through its effects on chromatin structure. Further, Myc is a key factor in induced pluripotency whereby fully differentiated cells can be converted to a pluripotent state by provision of four reprogramming factors (Sox2, Nanog, Oct4, and Myc). Although expression of Myc is required at various stages for correct development of normal hematopoietic lineages, this occurs under strict regulatory constraints. Sustained inappropriate over-expression of Myc leads, in many cases, to tumorigenesis. We have previously studied the ability of Myc to induce tumors in liver, osteosarcoma, and T-cell lymphoma transgenic mouse models. Here, we will focus on analysis of Myc-induced lymphomas, synergistically with our study of human lymphoid and myeloid malignancy. Our mouse model will enable us to analyze induction of self-renewal programs leading to tumor formation, in a controlled context with a specific mechanism of action (Myc over-expression). Using this system, we can also cause tumors to regress and then relapse, permitting analysis of the latter phenomenon and how it relates to cell states. Further, by manipulating genes such as P53, PI 6, Bim inactivation we will perturb processes of apoptosis and senescence to determine their effects on tumor onset and regression.

The targeted inactivation of specific oncogenes can result in the dramatic regression of cancers through the phenomenon of oncogene addiction (1-7). However, it is difficult to predict which tumors will exhibit addiction to a particular oncogene and which patients with cancer will respond to particular targeted therapies. We hypothesize that through the use of mathematical and statistical modeling together with quantitative molecular imaginq, we can predict verv early after the initiation of a therapv whether cancer patients will respond to tarqeted oncogene inactivation. As a direct outgrowth of our previous four years work as members of the Stanford ICMIC program, we have developed an approach to mathematically model tumorigenesis in conditional transgenic models of lung adenocarcinoma utilizing quantitative microCT imaging combined with in situ analysis. Our goal was to address two important and related questions: i) what is the biological mechanism governing the dramatic response of oncogene addicted tumors following oncogene inactivation and ii) how can we best predict which tumors are oncogene addicted versus which are not. To address these questions, we have made use of mechanistic modeling based on ordinary differential equations, together with data-driven statistical modeling based on support vector machine classifiers. Using a mechanistic model, we have found that oncogene inactivation in oncogene addicted tumors can be modeled as a differential attenuation of pro-survival and pro-death intracellular signals. Using a data-driven statistical model, we have been able to predict shortly after oncogene inactivation whether tumors are oncogene addicted or not. Moreover, we provide preliminary results showing that our predictive model can be applied to human patients (N=43) with lung tumors treated by EGFR inhibition with eriotinib, in order to predict both genotype and progression free survival. To date, we have focused on the use of anatomical imaging (microCT) and immunohistochemical analyses to provide the data for our models. However, anatomical imaging does not provide information on the biological activity of a tumor, while immunohistochemistry does not allow for serial sampling of a given tumor. We believe that we can now significantly improve our models and enhance their clinical transiational applicability for the analysis of human lung tumors by incorporating PET and SPECT imaging. In particular, we will incorporate molecular imaging of proliferation [FLT microPET] and apoptosis [ [99] Tc-AxV microSPECT] into our mechanistic model of oncogene addiction to replace immunohistochemical analyses, and into our predictive model of oncogene addiction status to improve the predictive power of the model. Furthermore, we will make use of a novel nanoscale proteomic technology, the Nano-lmmuno-Assay (NIA), for interrogating protein expression and phosphorylation in tumor samples (8). Thus, we will significantly enhance and extend our mathematical models and perform a prospective validation of our approach in a clinical study.

This is a new Research Program at the Stanford Cancer Center that has been established to promote collaborations among clinical and basic scientists to develop novel diagnostic and therapeutic approaches for cancer. The program consists of 41 members from 12 departments and 3 schools within the University and brings together chemists, biologists, statisticians and translational and clinical researchers with a common interest in the development of new cancer therapies. The Program has five major components: target identification and validation;drug discovery and delivery;mechanisms of drug action;molecular diagnostics;and clinical translational research. Research by program members has identified a novel synthetic chemistry approach to overcome taxol drug resistance, utilized carbon nanotechnology for the development of targeted delivery of Taxol (paclitaxel) or Doxorubicin, discovered that the inhibition of alpha PKC protects against breast cancer metastasis, defined genetic mechanisms for regulation of the MDR1/ABCB1 gene, developed a new technology for the nanoscale measurement of proteins for molecular diagnostics, applied high-throughput technologies to identify new agents that target the hedgehog pathway, and identified novel adenoviral based vectors for gene delivery to the liver. The Program is highly interactive through annual retreats, monthly research meetings, bi-annual faculty meetings, bi-annual student-sponsored symposia and invited speakers and symposia with pharmaceutical companies. The Program has catalyzed interactions leading to multi-investigator program awards including: a Leukemia and Lymphoma SCOR grant, a Department of Defense grant and an NIH Major Equipment Instrument Grant. Dr. Dean Felsher, Program Leader, is a physician scientist who is a leader in elucidating mechanisms of oncogene addiction through the use of transgenic animal models of lymphoma, hepatoma, osteosarcoma and lung adenocarcinoma. Dr. Branimir Sikic, Program Co-Leader, is a physician scientist whose research focuses on the pharmacology of drug resistance and who has been instrumental in implementing Phase I and II clinical trials. This program has the goal of conducting first-in- human Phase I trials of novel agents based on the best science.

DESCRIPTION (provided by applicant): The targeted inactivation of oncogene can elicit robust cell death suggesting that tumors can be addicted to a mutated proto-oncogene. Understanding why and predicting when tumors are addicted to oncogenes would have protean implications for the development of therapies for cancer. This would enable a more rational basis for choosing molecular targets, identify potentially new non-oncogene targets, facilitate the screening for potential agents and provide a strategy for selecting patients most likely to benefit from specific targeted therapeutic. To study oncogene addiction, we have used the Tet system to develop conditional transgenic mouse models. We have created several conditional oncogenes (MYC, RAS, BCL-2, BCR-ABL), to create transgenic mouse models of different cancers including: T-acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), osteogenic sarcoma (OS), and hepatocellular carcinoma (HCC). We have found that the consequences of oncogene inactivation are dependent upon both cellular and genetic context. Oncogene addiction involves tumor cell intrinsic and host dependent programs including the permanent loss of self- renewal or induction of cellular senescence and host-dependent programs. We are in the position to now define more generally how these mechanisms contribute oncogene inactivation induced cel death. Our hypothesis is that oncogene addiction can be modeled as a differential response between cel death and survival signaling. We will define key lynch pin gene products and pathways. Our approach will be to perform a quantitative in situ analysis using intravital microscopy and imunohistochemistry combined with a comparative proteomic and genomic analysis. We will perform these studies using different conditional oncogenes and types of cancer in different genetic contexts and then use mathematical modeling and computational biological approaches to reveal the common lynch pin genes and define their mechanistic role in oncogene addiction.

DESCRIPTION (provided by applicant): Renal cell carcinoma (RCC, kidney cancer) is one of the most commonly diagnosed cancers and is associated with significant mortality. In the United States in 2011, it is estimated that 60,920 patients will be diagnosed and 13,120 patients will die from kidney cancer. Data from the Surveillance, Epidemiology, and End Results program of the National Cancer Institute illustrates the steady rise of RCC incidence over the past 30 years. The majority of clear cell RCC patients have mutations in the von Hippel-Lindau (VHL) gene that result in unregulated expression of the hypoxia-inducible factor (HIF) transcription factor complex. Based on this recognition of the role of hypoxia in RCC carcinogenesis, most patients with metastatic RCC are treated with agents that target the HIF axis, either in the form of a VEGF or mTOR inhibitor. The VEGF inhibitors including small molecule multi-tyrosine kinase inhibitors (TKIs) such as sunitinib, pazopanib, sorafenib, and axitinib, and antibodies to VEGF such as bevacizumab have all shown clinical activity in RCC. Surprisingly however, despite the wide availability of these agents to treat RCC, many challenges remain: VEGF expression patterns and activation of downstream pathways in RCC are not well characterized. TKIs are non-selective and often incur significant toxic off-target effects that result in subtherapeutic treatment and futile toxicity in the 20% that do not respond. Thus, there is an urgent need to identify molecular markers that identify the relevant oncogenic pathway in each tumor to aid in the selection of systemic treatments and drastically reduce the amount of futile toxicity. However, identifying and developing biomarkers in RCC has been extremely challenging and a major deterrent has been the inability to measure specific biologic proteomic alterations in patient cells before, during and after treatment. Using existing methods, it is virtually impossibl to obtain real-time proteomic information from clinical specimens. We developed nanoscale immunoassay (NIA) as a highly sensitive, quantitative and unique way to concurrently measure proteins, their different isoforms and multiple phosphorylation states. Further, nanoscale proteomic analysis has the potential to dramatically reduce the amount of tissue required for diagnosis both in primary or other metastatic site biopsy. The ability to perform proteomic analysis of multiple tumor sites at a single time point from same patient, as well as in multiple samples over time, will accelerate translational research and ultimately improve care for patients with RCC. We believe that molecular markers hold the promise to inform every step of patient care in RCC including diagnosis, selection of surgical and systemic treatment, and monitoring response to targeted therapies. We aim to use NIA to identify distinct proteomic signatures of the commonly dysregulated hypoxia pathway in RCC patients and thus aid the selection of personalized and effective systemic therapies by targeting the patient's tumor-specific pathway(s). PUBLIC HEALTH RELEVANCE: Renal cell carcinoma (RCC, kidney cancer) is one of the most commonly diagnosed cancers and is associated with significant mortality. The treatment of RCC suffers from several critical issues that can be addressed using a novel nanoscale immunoassay (NIA), which is a highly sensitive and quantitative proteomic assay, for in vivo analysis of oncoproteins in scant clinical material. Our goal is to develop and validate NIA as a platform which can analyze clinical material from primary and metastatic RCC tumors and paired normal tissues, to discover proteomic biomarkers for diagnosis, treatment selection, and measures of therapeutic response.

DESCRIPTION (provided by applicant): Even when there is initial therapeutic sensitivity to a conventional chemotherapy or targeted therapy, tumors can become resistant and recur. Methods that model and predict therapeutic resistance of cancer can be extremely useful in the development of more effective treatments for cancer. Our long-term goal is to develop strategies to model, predict, and target therapeutic resistance of cancer. Our proposed approach is to utilize conditional transgenic mouse models combined with computational modeling. We hypothesize that therapeutic resistance to oncogene inactivation can be modeled and thus predicted as a consequence of clonal evolution of tumor cells driven by both cell autonomous and immune-mediated selective pressures. Our approach in Aim 1 is to build a mathematical model that incorporates the roles of the immune system and of evolutionary dynamics to predict the emergence of therapeutic resistance upon oncogene inactivation. Then, in Aim 2 we will experimentally interrogate the roles of the immune system and of evolutionary dynamics in the emergence of therapeutic resistance. We will directly examine immune effectors/cytokines and clonal evolution in our conditional transgenic mouse model with intravital microscopy and bioluminescence imaging. Finally, in Aim 3 we will validate in vivo our mathematical model's predictions of the emergence of therapeutic resistance under novel circumstances.

DESCRIPTION (provided by applicant): A hallmark feature of tumorigenesis is the global shift in metabolism. The ability to easily measure metabolism in situ could provide a powerful strategy for the early detection of cancers, as well as the ability to better diagnose and prognosticate cancers based on the detection of certain metabolic signatures related to specific oncogene expression and finally could uncover novel molecular underpinnings of cancer that could serve as better therapeutic targets. The MYC oncogene is one of the most commonly implicated causes of human tumorigenesis, in particular associated with the pathogenesis of hematopoietic tumors including lymphoma and epithelial tumors such as hepatocellular carcinoma. MYC contributes to tumorigenesis by functioning as a global regulator of transcription involving many cellular programs, in particular, cellular proliferation, growth and metabolism associated with the Warburg effect. MYC regulates key genes in glycolysis, glutaminolysis, tricarboxylic acid cycle, C1/folate, purine, pyrimidine and, notably, lipid metabolism. This suggests that in situ analysis of specific metabolic signatures could be a highly useful approach to detect, diagnose and prognosticate MYC-associated human tumors. However, using existing methods, it has not been readily possible to perform an in situ analysis of metabolism. Now, we have developed a highly sensitive approach that will enable us to use an innovative form of mass spectrometry to provide microscopic examination of the MYC-induced cancer metabolism. A key feature of this analysis is the ability to provide essentially rea time, in situ analysis. The method - called Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) - bombards cells and/or tissue sections with microdroplets containing acetonitrile and dimethylformamide that dissolve hundreds of lipids and metabolites. In this proposal, we will use DESI-MSI as a tool for identifying metabolic signatures causally associated with MYC expression in lymphomas. We will use this approach to decipher the biological mechanism by which MYC generates cancers thought the identified metabolites.

? DESCRIPTION (provided by applicant): The proposed postdoctoral training program, Cancer-Translational Nanotechnology Training (Cancer-TNT) Program, is a diverse and synergistic 3-year training program bringing together 25 faculty and 9 Departments from three schools to train the next generation of interdisciplinary leaders who will pursue challenges in cancer research and clinical translation. Cancer nanotechnology is a rapidly growing field that requires close interactions of researchers from disparate fields of science, such as chemistry, materials science, cancer biology, and medicine. During the proposed 5-year cycle, we will recruit a total of 12 postdoctoral trainees to provide them with education and cross-disciplinary training to develop interdisciplinary researchers in cancer nanotechnology translation. Eight (8) trainees will complete training during the 5-year cycle. Our trainees' skill sets will bridge multiple disciplines such as chemistry, molecular biology, bioengineering, molecular imaging, nanoengineering, and clinical cancer medicine. Trainees will be able to advance cancer research, diagnosis, and management. The Molecular Imaging Program at Stanford (MIPS) from the medicine side, with the Departments of Chemistry, Materials Science and Engineering from the engineering side, will provide a solid foundation for our program. The Stanford environment also leads a productive Center for Cancer Nanotechnology Excellence (CCNE-T), an NCI Designated Comprehensive Cancer Center, an In Vivo Cellular and Molecular Imaging Center (ICMIC @ Stanford), the highly effective interdisciplinary Bio-X program, facilities for Nanofabrication and Nanocharacterization, as well as Nanoinformatics capabilities, the Canary Center for Cancer Early Detection, and a Physical Sciences Oncology Center (PSOC), in which several of our mentors have leading roles. Through these activities, we have already been successfully training the next generation of cancer nanotechnologists and we are excited by the possibility to formalize and expand training activities through the Cancer-TNT. Our program includes a diverse group of faculty mentors representing five program areas. Through this mentor group, we are able to bring together formal courses in cancer biology, cancer immunology, molecular imaging, molecular pharmacology, and gene therapy, nanomedicine, micro/nanofabrication, biochips, electrical engineering, and materials science; hands-on training activities in Nanocharacterization; and a clinical component including Stanford Oncology Clinical Lecture Series. The Cancer-TNT fellows will be recruited into a 3-year program to complete coursework and research with two complementary mentors. Trainees will prepare a mock grant proposal in their second year to help them gain experience and confidence in the grant application process. A Training Committee will oversee trainee progress, with an Advisory Committee monitoring the entire program. Stanford University, with support from the NCI, is poised to be a major training center for the rapidly growing field of cancer nanotechnology.

ABSTRACT Tumorigenesis is associated with heterogeneous evolving changes of innate and adaptive immune effectors in the tumor microenvironment. Recording, identifying and quantifying these cellular and molecular changes could transform our understanding of tumorigenesis. We and others have shown that oncogenes, such as MYC co-opt and/or subvert these immune effectors, thereby evading immune detection and promoting a tumor microenvironment that fuels tumor growth; however, this appears to occur in a manner that necessarily and predictably leaves tumors highly vulnerable to acute oncogene inactivation, whereby immune effectors become activated, thereby resulting in dramatic tumor regression, a phenomenon called ?oncogene addiction?. Hence, we hypothesize that the identification?quantification and localization of these different host immune effectors in the tumor microenvironment during tumorigenesis and tumor regression will identify cellular biomarkers that will predict therapeutic response to oncogene inactivation. Our approach will be to employ the Tet system regulated model of MYC-induced hepatocellular carcinoma (HCC) and MYC/Twist1-induced model of metastatic HCC. Then, to utilize: FACS/CyTOF, CODEX/MIBI, IVM/BLI, and Gene expression/CIBERSORT to identify cellular effectors and hallmark genes and metabolites. First, our transgenic mouse model of MYC- induced HCC has been widely utilized by us and many others. Employing the Tet System, our model exhibit precise reversible and titrable control of the gene expression of the MYC oncogene. Tumor formation occurs slowly over time, oncogene-dependency results in tumor regression that is highly dependent on the immune response. Further, recently we generated a not published mouse model dramatically illustrating that MYC- induced liver tumors rapidly metastasize through the blood stream when combined with transgenic Twist1 expression. Twist1 has previously been associated with metastasis. We have obtained preliminary results that show metastasis is associated with and appears to require recruitment of macrophages. Finally, we have found that we can use human TCGA data available to identify potential cellular effectors associated with the pathogenesis of human HCC. Thus, all stages of tumorigenesis appear to be dependent on the stromal and immune response (initial, progressed, metastatic, regressed) and represent a unique tool to assess tumor- microenvironment heterogeneity. We have three aims: first, to dissect the mechanism by which the adaptive and innate immune system facilitates HCC progression, metastasis, and regression; second, to determine the kinetics and localizations of the above identified cell populations using in vivo (IVM and BLI) imaging technologies with special emphasis on cell-cell interactions and, third, to assess the ability of these findings to make predictions regarding the clinical behavior and prognosis of human HCC.

Abstract MYC is the most commonly activated oncogene in human cancer. However, to date, no existing therapies directly target MYC or the MYC pathway. My goal is now to target the MYC oncogene pathway to treat human cancer. Over the last 20 years, I have gained fundamental new insights into how the MYC oncogene initiates and maintains tumorigenesis. My work has established the idea that MYC is a hallmark of cancer and that many cancers are ?MYC oncogene addicted?. I have identified both tumor intrinsic and host-immune dependent mechanisms. Now, I will use these insights from lab and novel methods to develop new therapies for cancer. I was one of the first investigators to use the Tetracycline regulatory system (Tet system) to generate ?conditional? transgenic mouse models to demonstrate that MYC-induced cancer is ?reversible? or ?oncogene addicted? (Felsher and Bishop, Molecular Cell, 1999). Since then, I have used the Tet system to make a library of oncogene driven transgenic mouse models (MYC, RAS, BCR-ABL) of T-cell acute lymphoma (T-ALL), leukemia (AML), osteosarcoma (OS), hepatocellular carcinoma (HCC), lung adenocarcinoma (LAC) and renal cell adenocarcinoma (RCC). I have used my conditional transgenic mouse model systems to not only understand how MYC and other oncogenes initiate and maintain tumorigenesis but also develop innovative methods and novel technologies to make seminal contributions in cancer research, exhibiting sustained productivity. My proposed future research is built on recent observations that have used combined RNA, ChIP and metabolomic analysis to identify that lipogenesis and CRISPR synthetic lethal screen to identify nuclear transport as examples of otherwise not known to be MYC-regulated gene pathways that when targeted can block and reverse MYC- driven cancer. Now, I propose to use my library of conditional transgenic mouse models and human PDX models to generally identify targetable genes and pathways in the MYC oncogene pathway. I will use three complimentary approaches: RNAseq, ChIPseq and DESI-MSI to identify novel vulnerabilities in MYC-driven cancers; CRISPR in vitro and in vivo synthetic lethal screens combined with CyTOF and CODEX analysis to identify targets in my MYC-driven tumor models and understand their mechanistic role in tumorigenesis; MYC function reporter systems to be able to screen for genes and therapies to target MYC-driven cancers. My proposed research program has extensive support from an interdisciplinary team of colleagues. My proposed studies will glean novel mechanistic insights for how MYC drives tumorigenesis and use these insights to develop new therapeutic targets.